The nemaline myopathies are neuromuscular disorders characterized by muscle weakness and rod-shaped "nemaline" inclusions in skeletal muscle fibers Recent studies have identified a recessively inherited form of nemaline myopathy, the Amish nemaline myopathy (ANM), among the Old Order Amish at the rate of 1 of 500 births The molecular cause of ANM is a nonsense mutation in the gene encoding slow skeletal muscle troponin T (TnT), a muscle- , , 2+ specific Ca -regulatory protein truncating the protein at amino acid 179 We have shown that the truncated slow TnT is degraded and not incorporated into the myofibrils, consistent with the recessive inheritance of the disease The ANM phenotypes include tremors, contractures and hypotonia A rapid postnatal progress results in death from respiratory insufficiency, usually in the second year No effective treatment is available The discovery of ANM gene has given the affected families the first hope for a cure of this fatal disease Three homologous genes are present in the vertebrate genome encoding the slow, fast and cardiac TnT isoforms Despite the loss of slow TnT in ANM muscle, only mild phenotypes are shown at birth The postnatal worsening of myopathy is concurrent with the developmental down-regulation of cardiac TnT in skeletal muscle and the embryonic to adult isoform transition of fast skeletal muscle TnT Therefore, cardiac and embryonic fast TnT, but not adult fast TnT, may compensate for the lost function of slow TnT To understand the pathology of ANM in which the loss of only one isoform of TnT causes severe myopathy, we shall investigate the functional relationship between TnT isoforms In addition to a better understanding of the CaZ*-regulation of muscle contraction this study aims at the development of therapies for ANM Four specific aims are proposed to bridge tile basic studies of TnT isoform gene regulation and structure-function relationships to the clinical management of this lethal disease I Biochemical characterization of TnT iscforms to investigate their functional difference and whether the cardiac TnT and embryonic fast TnT, both of which are acidic isoforms, are functionally similar to slow TnT which is also acidic II Functional characterization of the truncated slow TnT to determine whether it may have a dominant negative effect in ANM II1 Protein interaction and transgenic expression experiments to investigate whether disorganization of muscle thin filament due to change in one regulatory protein forms the basis of nemaline myopathy IV Production and characterization of the slow TnT mutation in an animal model of ANM to study muscle pathophysiology, fatigue tolerance, and therapeutic approaches PERFORMANCESITE(S) (organizationcity state) Case Western Reserve University, Cleveland, Ohio KEYPERSONNEL Seeinstructions Usecontinuatiopnagesasneededto providetherequiredinformatiointhe formatshownbelow StartwithPrincipaIlnvestigatorListallother keypersonnelin alphabeticaolrder last namefirst Name Organization Roleon Project J -P Jin Case Western Reserve University Principal Investigator Q -Q Huang Case Western Reserve University Research Associate O Ogut Case Western Reserve University Postdoctoral Fellow B Biesiadecki Case Western Reserve University Graduate Student T M Nosek Case Western Reserve University Co-Investigator C Colmenares Cleveland Clinic Foundation Consultant T O Crawford Johns Hopkins University Consultant D H Morton Clinic for Special Children Consultant DisclosurePermissionStatement Applicableto SBIR/STTROnly. Sea instructions[] Yes [] No _ PHS398 (Rev 05/01) Page2 FormPage2 _ Principal Investigator/Program Director (Last first middle) JIN_ J,-P. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,